Solution for injection and preparation method thereof

Abstract

The invention relates to the technical field of medical treatment and beauty treatment, in particular to a solution for injection and a preparation method thereof. According to the injection solution disclosed by the invention, the cross-linked sodium hyaluronate, the vitamin B, the active peptide, the nicotinamide, the amino acid, the buffer solution and the water for injection are taken as raw materials, so that the injection solution is endowed with relatively good enzyme stability, is not easy to quickly decompose in vivo and is non-toxic; functional raw materials such as active peptide and nicotinamide are used, so that the injection solution has excellent anti-aging and melanin inhibition effects.

Description

Technical Field

[0001] The present invention relates to the field of medical and cosmetic technology, and in particular to an injection solution and a preparation method thereof. Background Art

[0002] Liquid injection, also known as injection, commonly known as "water injection", is a preparation made by preparing the drug into a solution (aqueous or non-aqueous), suspension or emulsion, and filling it into a container. It is mainly determined by the nature of the drug and the medical requirements. Generally, water-soluble drugs are required to achieve rapid effects after injection, so they are mostly prepared into aqueous solutions or water-complex solutions (such as adding ethanol, propylene glycol, glycerol, etc. to the aqueous solution). Some drugs are not suitable for aqueous solutions, such as those that are difficult to dissolve in water or to prolong the efficacy after injection. They can be made into oil solutions, water or oil suspensions, and emulsions. However, these injections are generally only for intramuscular injection.

[0003] At present, injections containing sodium hyaluronate are common medical or cosmetic injections. Injections containing sodium hyaluronate are widely used in animal tissues and body fluids, and usually can moisturize and replenish water, and can also moisturize the skin. If the skin has symptoms such as itching and redness caused by allergies, sodium hyaluronate injections can also be used for treatment, which can improve the symptoms. At the same time, it can also improve joint pain and joint diseases caused by knee joint injuries.

[0004] However, this type of injection often suffers from enzyme instability, is easily broken down quickly by enzymes in the body, has poor effects, and has relatively simple functions. Summary of the invention

[0005] The present invention aims at the above technical problems and proposes a technical solution of the present invention, which includes an injection solution and a preparation method thereof.

[0006] The injection solution of the present invention adopts cross-linked sodium hyaluronate, vitamin B, active peptides, nicotinamide, amino acids, buffer solution and water for injection as raw materials, which endows the injection solution with good enzyme stability, is not easy to be rapidly decomposed in the body, and is non-toxic; the use of functional raw materials such as active peptides and nicotinamide makes the injection solution have excellent anti-aging and melanin inhibition effects.

[0007] Furthermore, the raw material composition of the injection solution of the present invention is: 10-50g / L cross-linked sodium hyaluronate, 0.5-3mg / L vitamin B, 0.1-2g / L active peptide, 0.1-1g / L niacinamide, 200-950mg / L amino acids and the balance water for injection.

[0008] Furthermore, the active peptide includes at least one of decapeptide-4, acetyl tetrapeptide-5, and glutathione.

[0009] Furthermore, in order to enhance the anti-aging effect, it is desirable to select a mass ratio of decapeptide-4 to acetyl tetrapeptide-5 of 1:2-4. The total concentration of decapeptide-4 and acetyl tetrapeptide-5 in the injection solution is 0.5-1 g / L.

[0010] Furthermore, the amino acid includes at least one of proline, glycine, alanine and leucine.

[0011] Furthermore, the preparation method of the cross-linked sodium hyaluronate includes adjusting the pH of hyaluronic acid, then adding calcium chloride and 1,2-cyclohexanediol diglycidyl ether to stir and react, drying, crushing, adjusting the pH for the second time, adding buffer solution for dialysis, drying, and crushing to obtain the cross-linked sodium hyaluronate.

[0012] Furthermore, the molecular weight of the hyaluronic acid is 1.2 million to 1.7 million Da.

[0013] Furthermore, the mass ratio of calcium chloride to 1,2-cyclohexanediol diglycidyl ether is 1:3-5.

[0014] Furthermore, the specific preparation method of the cross-linked sodium hyaluronate comprises the following steps:

[0015] Step 1: Mix hyaluronic acid and water in a mass ratio of 1:20-40, and adjust the pH to 5.8-6.2 to obtain a mixture 1;

[0016] Step 2: Add calcium chloride and 1,2-cyclohexanediol diglycidyl ether in a mass ratio of 1:3-5 to the mixture 1, heat to 40-55° C., stir for 5-10 hours, and dry to obtain a dried product; wherein the total mass of the calcium chloride and 1,2-cyclohexanediol diglycidyl ether is 1-6% of the mass of the hyaluronic acid;

[0017] Step 3: The dried product is crushed and mixed with water at a mass ratio of 1:12-25, and the pH is adjusted to 6.7-7.2 for a second time, and then phosphate buffer is added for dialysis, and the dialyzed liquid is removed to obtain a concentrate, and the concentrate is freeze-dried and crushed to obtain the cross-linked sodium hyaluronate.

[0018] Furthermore, the drying in step 2 is vacuum drying, the vacuum drying temperature is 45-52° C., the drying time is 6-15 hours, and the drying pressure is 0.02-0.05 MPa.

[0019] Furthermore, based on the functional considerations of the injection solution, cell lyophilized powder can be added. The cells are derived from at least one of amniotic membrane, umbilical cord or placenta, and the amount of cell lyophilized powder in the injection solution is 0.005-0.5 g / L. The amniotic membrane, umbilical cord or placenta can be derived from humans and / or animals.

[0020] The present invention also provides a method for preparing the injection solution, comprising the step of mixing the raw materials with injection water.

[0021] The injection solution of the present invention belongs to medicines and can be used in medicines and drugs, including chemical drugs or biological drugs.

[0022] The injection solution of the present invention belongs to a cosmetic product and can be used in cosmetic products.

[0023] The injection solution of the present invention can also be used for cosmetic purposes, specifically, it can be used to prepare hyaluronic acid injection products.

[0024] Beneficial effects of the present invention:

[0025] The injection solution of the present invention adopts cross-linked sodium hyaluronate, vitamin B, active peptides, nicotinamide, amino acids, buffer solution and water for injection as raw materials, which endows the injection solution with good enzyme stability, is not easy to be rapidly decomposed in the body, and is non-toxic; the use of functional raw materials such as active peptides and nicotinamide makes the injection solution have excellent anti-aging and melanin inhibition effects.

[0026] The relative proliferation rate RCR value of cells in the cross-linked sodium hyaluronate dilution prepared by the present invention reaches 89.64%, and the in vitro anti-hyaluronidase decomposition coefficient R value reaches 85.49%; the cross-linked sodium hyaluronate prepared by the present invention has low cytotoxicity, good safety performance, excellent in vitro anti-hyaluronidase decomposition performance, and can stably exist in the body.

[0027] The present invention uses calcium chloride and 1,2-cyclohexanediol diglycidyl ether to give cross-linked sodium hyaluronate better low toxicity effect and enzyme stability. Compared with other cross-linking agents, the cross-linking agent selected in the present invention can bring better effects.

[0028] The calcium chloride and 1,2-cyclohexanediol diglycidyl ether used in the present invention have a mutually reinforcing relationship in imparting low toxicity and stability to cross-linked sodium hyaluronate, and the two synergistically enhance the safety and stability of cross-linked sodium hyaluronate. The effect produced is better when the ratio of the two is 1:3-5.

[0029] The decapeptide-4 and acetyl tetrapeptide-5 used in the present invention both have excellent functions of increasing the anti-aging effect of the product. When the two are used in combination, the effect is better, especially when the mass ratio of the two is 1:2-4 for decapeptide-4 and acetyl tetrapeptide-5, the increased skin elasticity and the anti-aging effect of the skin are very good.

[0030] The injection solution prepared by the invention has the effects of being odorless and easy to squeeze, and also has the effects of low heavy metal content, low bacterial endotoxin EU content, low toxicity and low allergenicity. DETAILED DESCRIPTION

[0031] The present invention will be described below in conjunction with specific embodiments, and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments are used to illustrate the present invention, rather than to limit the present invention.

[0032] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0033] 1. Preparation and effect verification of cross-linked sodium hyaluronate

[0034] 1. Cross-linked Sodium Hyaluronate A

[0035] The preparation method is as follows:

[0036] Step 1: mixing hyaluronic acid with a molecular weight of 1 million Da with deionized water at a mass ratio of 1:30, and adjusting the pH to 5.92 using sodium hydroxide to obtain a mixture 1;

[0037] Step 2: Add calcium chloride and 1,2-cyclohexanediol diglycidyl ether in a mass ratio of 1:4.5 to the mixture 1, heat to 47.5°C, stir for 8 hours, and dry for 10 hours at a pressure of 0.04 MPa and a temperature of 48°C to obtain a dried product;

[0038] Wherein, the total mass of calcium chloride and 1,2-cyclohexanediol diglycidyl ether in step 2 is 4.57% of the mass of hyaluronic acid;

[0039] Step 3: The dried product is crushed to a size that can pass through a 20-mesh sieve and mixed with deionized water at a mass ratio of 1:20. Sodium hydroxide is added for a second time to adjust the pH to 6.9 to obtain an adjusted solution. A phosphate buffer solution with a pH of 6.92 of 5 times the mass of the adjusted solution is added to mix and dialyze. The dialysate is removed and the obtained concentrate is mixed and dialyzed again using 20 times the mass of a phosphate buffer solution with a pH of 6.92. This is repeated twice. The dialyzed liquid is removed to obtain a concentrate. The concentrate is freeze-dried at -40°C to a water content of 4.37wt%, and crushed to obtain the cross-linked sodium hyaluronate.

[0040] 2. Cross-linked sodium hyaluronate B

[0041] The preparation method is as follows:

[0042] Step 1: mixing hyaluronic acid with a molecular weight of 1 million Da and deionized water in a mass ratio of 1:35, and adjusting the pH to 6.1 using sodium hydroxide to obtain a mixture 1;

[0043] Step 2: Add calcium chloride and 1,2-cyclohexanediol diglycidyl ether in a mass ratio of 1:3 to the mixture 1, heat to 45°C, stir for 7.5 hours, and dry for 8.7 hours at a pressure of 0.04 MPa and a temperature of 47°C to obtain a dried product;

[0044] Wherein, the total mass of calcium chloride and 1,2-cyclohexanediol diglycidyl ether in step 2 is 4.62% of the mass of hyaluronic acid;

[0045] Step 3: The dried product is crushed to a size that can pass through a 20-mesh sieve and mixed with deionized water at a mass ratio of 1:25, and sodium hydroxide is added for a second time to adjust the pH to 6.85 to obtain an adjusted solution, and then a phosphate buffer solution with a pH of 6.85 of 4 times the mass of the adjusted solution is added to mix and dialyze, the dialysate is removed and the obtained concentrate is mixed and dialyzed again using 20 times the mass of a phosphate buffer solution with a pH of 6.85, and the mixture is repeated twice, and then the dialyzed liquid is removed to obtain a concentrate, and the concentrate is freeze-dried at -40°C to a water content of 4.29wt%, and pulverized to obtain the cross-linked sodium hyaluronate.

[0046] 3. Cross-linked Sodium Hyaluronate C

[0047] Step 2 is replaced by: adding deionized water with a mass percentage of 4.57% of hyaluronic acid to mixture 1, heating to 47.5°C, stirring for 8 hours, and drying at a pressure of 0.04 MPa and a temperature of 48°C for 10 hours to obtain a dried product. The rest is the same as that of cross-linked sodium hyaluronate A.

[0048] 4. Cross-linked sodium hyaluronate D

[0049] Step 2 is replaced by: Step 2: Add calcium chloride to mixture 1, heat to 47.5°C, stir for 8 hours, and dry for 10 hours at a pressure of 0.04 MPa and a temperature of 48°C to obtain a dried product; wherein the mass of calcium chloride in step 2 is 4.57% of the mass of hyaluronic acid. Others are the same as those of cross-linked sodium hyaluronate A.

[0050] 5. Cross-linked Sodium Hyaluronate E

[0051] Step 2 is replaced by: adding 1,2-cyclohexanediol diglycidyl ether to mixture 1, heating to 47.5°C, stirring for 8 hours, and drying for 10 hours at a pressure of 0.04 MPa and a temperature of 48°C to obtain a dried product; wherein the mass of 1,2-cyclohexanediol diglycidyl ether in step 2 is 4.57% of the mass of hyaluronic acid. Others are the same as those of cross-linked sodium hyaluronate A.

[0052] 6. Cross-linked Sodium Hyaluronate F

[0053] Step 2 is replaced by: adding calcium chloride and 1,2-cyclohexanediol diglycidyl ether in a mass ratio of 1:1 to mixture 1, heating to 47.5°C, stirring for 8 hours, and drying for 10 hours at a pressure of 0.04 MPa and a temperature of 48°C to obtain a dried product; wherein the total mass of calcium chloride and 1,2-cyclohexanediol diglycidyl ether in step 2 is 4.57% of the mass of hyaluronic acid. Others are the same as those of cross-linked sodium hyaluronate A.

[0054] 7. Cross-linked Sodium Hyaluronate G

[0055] Step 2 is replaced by: adding calcium chloride and 1,2-cyclohexanediol diglycidyl ether in a mass ratio of 1:8 to mixture 1, heating to 47.5°C, stirring for 8 hours, and drying for 10 hours at a pressure of 0.04 MPa and a temperature of 48°C to obtain a dried product; wherein the total mass of calcium chloride and 1,2-cyclohexanediol diglycidyl ether in step 2 is 4.57% of the mass of hyaluronic acid. Others are the same as those of cross-linked sodium hyaluronate A.

[0056] 8. Cross-linked sodium hyaluronate H

[0057] Step 2 is replaced by: adding glycerol triglycidyl ether to mixture 1, heating to 47.5°C, stirring for 8 hours, and drying for 10 hours at a pressure of 0.04 MPa and a temperature of 48°C to obtain a dried product; wherein the mass of glycerol triglycidyl ether in step 2 is 4.57% of the mass of hyaluronic acid. Others are the same as those of cross-linked sodium hyaluronate A.

[0058] The cross-linked sodium hyaluronate was subjected to the following performance tests. Before the test, the cross-linked sodium hyaluronate prepared above was diluted to a concentration of 15 g / L with a phosphate buffer having a pH of 7, and sheared at a high speed of 10°C and 5000 rpm for 1 hour, passed through a 300 mesh sieve, and filtered to obtain a filtered liquid, which was sterilized and then used as a sample for testing.

[0059] Cytotoxicity: Refer to "Biological Evaluation of Medical Devices - In Vitro Cytotoxicity Test" to detect cell proliferation rate. According to the extraction method, L929 cells were tested for cytotoxicity, the absorbance value was measured using an enzyme-linked immunosorbent assay, and the relative proliferation rate RCR value of the cells was calculated; the higher the RCR, the better the biocompatibility and the lower the toxicity. The specific test results are shown in Table 1.

[0060] Enzyme stability: Take 0.5g of the above filtered liquid, add 2mL of 300U / mL hyaluronidase solution, and enzymolyze at 37℃ for 45 hours; add PBS buffer to 5mL, take 1mL of it and add 4mL of anhydrous ethanol to mix evenly, centrifuge at 10000r / min for 12min, take 2mL of supernatant and dilute to 5mL with PBS buffer as A1 liquid. Take 0.5g of the above filtered liquid, add 10mL of 0.5mol / L sulfuric acid aqueous solution, hydrolyze in boiling water bath for 15min, dilute to 100mL with deionized water as A2 liquid. Take 1mL of A1 liquid and A2 liquid respectively, and measure the glucuronic acid content in A1 liquid and A2 liquid by improved carbazole colorimetric method. Calculate the in vitro anti-hyaluronidase decomposition coefficient R of the liquid, R=100%×(1-0.625α1 / α2), α1 is the glucuronic acid content in A1 liquid, and α2 is the glucuronic acid content in A2 liquid. The higher the in vitro anti-hyaluronidase decomposition coefficient R, the better the anti-hyaluronidase decomposition performance, the less rapid decomposition of cross-linked sodium hyaluronate in the human body, and the longer the storage time. The results of the enzyme stability test are shown in Table 1.

[0061] Table 1: Cytotoxicity and enzyme stability test results

[0062]

[0063]

[0064] It can be seen from the test results in Table 1 that the relative proliferation rate RCR value of the cells in the cross-linked sodium hyaluronate dilution prepared by the present invention reaches 89.64%, and the in vitro anti-hyaluronidase decomposition coefficient R value reaches 85.49%; the cross-linked sodium hyaluronate prepared by the present invention has low cytotoxicity, good safety performance, excellent in vitro anti-hyaluronidase decomposition performance, and can stably exist in the body.

[0065] By comparing cross-linked sodium hyaluronate A with C and H, it can be seen that the present invention uses calcium chloride and 1,2-cyclohexanediol diglycidyl ether to give cross-linked sodium hyaluronate better low toxicity effect and enzyme stability. Compared with other cross-linking agents, the cross-linking agent selected in the present invention can bring better effects.

[0066] The test results of cross-linked sodium hyaluronate A, D, and E show that calcium chloride and 1,2-cyclohexanediol diglycidyl ether have a mutually reinforcing relationship in imparting low toxicity and stability to cross-linked sodium hyaluronate, and the two synergistically enhance the safety and stability of cross-linked sodium hyaluronate. The effect produced by the ratio of the two is 1:3-5.

[0067] 2. Selection and effect verification of active peptides

[0068] Active peptides were diluted with deionized water to prepare active peptide solutions; the specific method was to dilute the active peptides with deionized water to a total concentration of 0.8 g / L to obtain active peptide solutions, and test the anti-aging effects of active peptide solutions prepared from different active peptides. Among them, the active peptides were decapeptide-4, acetyl tetrapeptide-5, or a combination of the two in different proportions. The active peptide solutions were then tested for anti-aging effects.

[0069] The specific test method is: volunteers with healthy skin and aged 30-45 years old were selected and randomly divided into groups with 6 volunteers in each group. Each group of volunteers used 0.7g of the above-mentioned active peptide solution and applied it evenly on the face every night for 50 consecutive days. The MPA580 test instrument was used to test the skin elasticity value R2 before and after 50 days of use of the active peptide solution; among them, R2 represents the ratio of the rebound amount of the skin without negative pressure to the maximum stretching amount under negative pressure. The closer to 1, the better the elasticity, ranging from 0 to 1. Then calculate the degree of change in the skin elasticity value R2 before and after using the active peptide solution for 50 days. The test results are shown in Table 2.

[0070] Table 2: Anti-aging effect test

[0071]

[0072]

[0073] According to the test content in Table 2, both decapeptide-4 and acetyl tetrapeptide-5 have excellent functions of increasing the anti-aging effect of the product. When the two are used together, the effect is better, especially when the mass ratio of decapeptide-4 to acetyl tetrapeptide-5 is 1:2-4, the increase in skin elasticity and the anti-aging effect on the skin are very good.

[0074] 3. Preparation and effect testing of injection solution

[0075] Solution 1 for injection: Raw materials: 15 g / L cross-linked sodium hyaluronate A, 2 mg / L vitamin B, 0.2 g / L decapeptide-4, 0.6 g / L acetyl tetrapeptide-5, 0.1 g / L glutathione, 0.2 g / L niacinamide, 100 mg / L proline, 50 mg / L glycine, 150 mg / L alanine, 150 mg / L leucine and the balance water for injection.

[0076] Solution 2 for injection: Raw materials: 15 g / L cross-linked sodium hyaluronate B, 1.5 mg / L vitamin B, 0.22 g / L decapeptide-4, 0.58 g / L acetyl tetrapeptide-5, 0.15 g / L glutathione, 0.15 g / L niacinamide, 150 mg / L proline, 150 mg / L glycine, 50 mg / L alanine, 50 mg / L leucine and the balance water for injection.

[0077] Solution for injection 3: Raw materials: 15g / L cross-linked sodium hyaluronate A, 2mg / L vitamin B, 0.2g / L decapeptide-4, 0.6g / L acetyl tetrapeptide-5, 0.1g / L glutathione, 0.2g / L nicotinamide, 100mg / L proline, 50mg / L glycine, 150mg / L alanine, 150mg / L leucine, 0.02g / L cell disruptor lyophilized powder and the balance water for injection;

[0078] The preparation method of cell disruption freeze-dried powder is as follows:

[0079] Step S1, washing and disinfecting the umbilical cord (human source) tissue, then breaking it, inoculating it into a culture medium (culture medium composition: DMEM / F12 culture medium + glutamine 12 μg / mL + insulin 20 μg / mL + dextran 12 μg / mL + glucose 0.05 mg / mL) for subculture, and collecting the cell culture supernatant of the 13th generation;

[0080] Step S2, adding 1.5% vitamin C by weight of the supernatant to the supernatant of the cell culture of the 13th generation and stirring evenly, then adding 0.085% sodium chloride by weight of the supernatant and stirring evenly; treating with an ultrasonic cell disruptor at 20°C for 10 min, with a processing power of 500 W and a frequency of 24 kHz, and then centrifuging at 3000 rpm for 15 minutes, and freeze-drying the supernatant to a water content of about 3.17 wt% to obtain a freeze-dried powder of the cell disruptor.

[0081] The preparation method of the injection solution comprises the steps of mixing the raw materials with injection water at 60 rpm for 0.5 hours, then filtering through 300 meshes, and sterilizing the filtrate to obtain the injection solution.

[0082] The above solution for injection was subjected to performance test, and the test results are shown in Table 3.

[0083] Table 3: Performance test of injection solution

[0084] performance Solution for injection1 Solution for injection2 Solution for injection3 odor No odor No odor No odor Heavy metal μg / g ＜5 ＜5 ＜5 Bacterial endotoxin EU / mL ＜0.05 ＜0.05 ＜0.05 Extrusion force N ＜30 ＜30 ＜30 Cytotoxicity assay ≤ Grade I ≤ Grade I ≤ Grade I Skin sensitization test ≤ Grade I ≤ Grade I ≤ Grade I

[0085] It can be seen from the test in Table 3 that the injection solution of the present invention has the effects of being odorless and easy to squeeze, and also has the effects of low heavy metal content, low bacterial endotoxin EU content, low toxicity and low allergenicity.

[0086] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A solution for injection, characterized in that: The preparation raw materials include cross-linked sodium hyaluronate, vitamin B, active peptides, niacinamide, amino acids, buffer solution and water for injection.

2. The solution for injection according to claim 1, characterized in that The raw material composition of the injection solution is: 10-50g / L cross-linked sodium hyaluronate, 0.5-3mg / L vitamin B, 0.1-2g / L active peptide, 0.1-1g / L niacinamide, 200-950mg / L amino acid and the balance of water for injection.

3. The solution for injection according to claim 1, characterized in that It also includes freeze-dried powder of cell disruptors, wherein the cells are derived from at least one of amniotic membrane, umbilical cord or placenta, and the amount of freeze-dried powder of cell disruptors in the injection solution is 0.005-0.5 g / L.

4. The solution for injection according to claim 2, characterized in that: The active peptides include at least one of decapeptide-4, acetyl tetrapeptide-5 and glutathione.

5. The solution for injection according to claim 2, characterized in that: The amino acids include at least one of proline, glycine, alanine and leucine.

6. The solution for injection according to claim 3, characterized in that: The mass ratio of decapeptide-4 to acetyl tetrapeptide-5 is 1:2-4, and the total concentration of decapeptide-4 and acetyl tetrapeptide-5 in the injection solution is 0.5-1 g / L.

7. The solution for injection according to claim 2, characterized in that: The preparation method of the cross-linked sodium hyaluronate comprises adjusting the pH of hyaluronic acid, then adding calcium chloride and 1,2-cyclohexanediol diglycidyl ether to stir and react, drying, crushing, adjusting the pH for the second time, adding buffer solution to perform dialysis, drying, and crushing to obtain the cross-linked sodium hyaluronate.

8. The solution for injection according to claim 7, characterized in that: The molecular weight of hyaluronic acid is 1.2-1.7 million Da.

9. The solution for injection according to claim 7, characterized in that: The mass ratio of calcium chloride to 1,2-cyclohexanediol diglycidyl ether is 1:3-5.

10. The method for preparing the injection solution according to any one of claims 1 to 9, characterized in that: The method comprises the steps of mixing the raw materials with water for injection.

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